Lithium acetate

Strains for in vivo analyses of Faa1 localization and function derive from genetic modification of S. cerevisiae w303. Knockouts and tags were obtained via homologous recombination of PCR products of interest. Briefly, log-phase cells were washed with lithium acetate (100 mM) and incubated in a mix containing lithium acetate (L6883; Sigma-Aldrich), PEG 2000 (202509; Sigma-Aldrich), salmon sperm DNA (15632011; Invitrogen), and the PCR product of interest at 30°C for 30 min and then shifted to 42°C for 20 min.
Strains were grown in a synthetic complete medium (0.17% [wt/vol] yeast nitrogen base without amino acids and ammonium sulfate [233520; BD] supplemented with 0.5% [wt/vol] ammonium sulfate [31119-M; Sigma-Aldrich], 2% [wt/vol] a-D-glucose monohydrate [16301; Sigma-Aldrich], and complete supplement [CSM] drop out [DCS0019; Formedium]). Log-phase cells were harvested, washed five times, and resuspended in SD-N medium (0.17% [wt/vol] yeast nitrogen base without amino acids and ammonium sulfate [233520; BD] and 2% [wt/vol] a-D-glucose monohydrate [16301; Sigma-Aldrich]). Based on the experiment, the SD-N medium was supplemented with cerulenin (219557, 50 µg/ml; Sigma-Aldrich) or glucose was reduced to 0.01% (wt/vol). Rapamycin (553210, 400 ng/ml; Calbiochem) was added to the synthetic complete medium.

Cadmium oxide (CdO, 99.99%, trace metals), trioctylphosphine (TOP, 90% technical grade), sulfur (S, 99.98%), 1-octadecene (ODE, 90%, technical grade), iso-Octane (99.7%, HPLC grade), 1-Octanethiol (> 98.5%), trimethylammonium chloride (TMACl, > 98%), potassium hydroxide (KOH, 99.99%), dimethyl sulfoxide (DMSO, > 99.9%) magnesium acetate tetrahydrate (99%), zinc acetate dihydrate (> 98%), lithium acetate (99.95%) and 1-butanol (anhydrous, 99.8%) were purchased from Sigma-Aldrich. Zinc acetate anhydrous (+ 99.9%) and ethyl acetate (> 99.5%, ACS certified) were purchased from Thermoscientific. Selenium (Se, 99.999%, metals basis) and oleic acid (90%, technical grade) were purchased from Alfa-Aesar. Octane (+ 99%, extra pure) was purchased from Acros Organics. All the reagents were used as received. Poly(ethylene dioxythiophene): polystyrene sulfonate (PEDOT: PSS) was purchased from Ossila. Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(4,4’-(N-(p-butylphenyl)) diphenylamine)] (TFB) was purchased from American Dye Source. Polyvinylpyrrolidone (PVP10) with an average molecular weight of 10,000 was purchased from Sigma-Aldrich. Patterned ITO-glass substrates with 15 Ω resistance and 25.4 mm × 25.4 mm × 0.7 mm were purchased from Luminescence Technology Corp.

We used the Sb competent cell preparation and transformation protocol from [24 (link)], which is based on the protocol from [33 (link)]. After transformation, the cells were plated on appropriate growth/selection media. Sb strains cultured overnight in appropriate media (Yeast Extract–Peptone–Dextrose (YPD) or Yeast Synthetic Drop-out) in a shaking incubator (37 °C, 250 rpm). Saturated cultures were subinoculated to pre-warmed media at starting OD 0.25 and grown for 3–4 h to OD600 1.0 (37 °C, 250 rpm). Cells were then harvested at 3000g for 5 min at room temperature and washed twice with sterile water and third time before with 100 mM Lithium Acetate. Supernatant was then removed. Then, solutions were added to the cells in the following order: 260 μL 50% PEG3350 (Fisher Scientific), 36 μL 1 M Lithium Acetate (Sigma-Aldrich), 50 μL of 2 mg/mL single-stranded salmon sperm DNA (Invitrogen, 15632011), 0.1–10 μg DNA. The mixture was then gently vortexed and then incubated at 42 °C for 1 h. This mixture was then centrifuged at 3000g for 1 min and the supernatant was discarded. The cell pellet was resuspended in 1 mL YPD by gently pipetting up and down and this tube was placed at a shaking incubator (37 °C, 250 rpm) for 1 h. Then, the cell suspension was centrifuged for 1 min at 3000g, resuspended in 25 μL sterile water, and plated on an appropriate growth media.